The long term goal of this program is to identify new factors that affect glycosylation, and to determine how they impact the functional mammalian glycome. Under the auspices of this grant, we discovered a novel inhibitor of MGAT1/GlcNAcT-I, the transferase that initiates the synthesis of complex N-glycans. The inhibitor is GlcNAcT-I Inhibitory Protein (GnT1IP) and the membrane- bound form, GnT1IP-L, is the physiological inhibitor of MGAT1. When the synthesis of complex N- glycans is inhibited by GnT1IP-L, N-glycan sites are solely occupied by high mannose N-glycans. Importantly, the GnT1IP gene is robustly expressed in testis, and reduced expression correlates with infertility syndromes in humans. Humans and rodents express GnT1IP-L in testis. In male mice, GnT1IP-L transcripts are found in spermatocytes, but not in spermatids. Finally, GnT1IP-L expression and inhibition of MGAT1 activity markedly enhance cell adhesion to Sertoli cells. We hypothesize that GnT1IP-L functions in spermatocytes to specifically inhibit MGAT1, thereby allowing spermatocytes to associate appropriately with Sertoli cells and spermatogenesis to proceed. Interestingly, conditional deletion of Mgat1 in spermatogonia blocks spermatogenesis beyond the spermatocyte stage, causing spermatids to form multinucleated giant cells. Therefore, we hypothesize that complex N-glycans are required in spermatids for differentiation into spermatozoa. We will test both hypotheses directly, and also determine the mechanism by which GnT1IP-L acts to target and inactivate MGAT1. Specific Aim 1 will test the hypothesis that GnT1IP-L acts specifically in the kin recognition complex that resides in the medial Golgi and contains MGAT1, MGAT2, MAN2A1 and MAN2A2. We will identify members of the GnT1IP-L complex, and whether GnT1IP-L inhibits MGAT1 directly in insect cells or in vitro. If soluble inhibitory GnT1IP-L/MGAT1 complexes can be made, they will be analyzed by X-ray crystallography. Specific Aim 2 will test the hypothesis that GnT1IP-L functions in spermatocytes to enable spermatogenesis. The consequences of misexpressing Mgat1 in spermatocytes to overwhelm the inhibitory activity of GnT1IP-L, and of conditionally deleting GnT1IP-L in spermatogonia or spermatocytes will be determined. Glycoprotein targets of GnT1IP-L in spermatocytes will be identified by a novel strategy. Human GnT1IP-L and MGAT1 transcripts will be characterized in human testis biopsies of males with and without infertility syndromes. Specific Aim 3 will test the hypothesis that MGAT1 is required for the differentiation of spermatids to form spermatozoa. Loss of MGAT1 blocks spermatogenesis due to fusion of spermatids. We will determine if expression of MGAT1 in spermatids or spermatocytes lacking MGAT1 rescues the block, and if conditional knockout of MGAT1 in spermatocytes gives the same phenotype. The molecular basis of the requirement for MGAT1 will be investigated by candidate and unbiased approaches.